15th European Molecular Imaging Meeting
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PET/SPECT, Radionuclide, X-Ray, CT I | Probe Chemistry

Session chair: Sam Bayat (Grenoble, France); Sara Lacerda (Orleans, France)
 
Shortcut: PW10
Date: Wednesday, 26 August, 2020, 5:30 p.m. - 7:00 p.m.
Session type: Poster

Contents

Abstract/Video opens by clicking at the talk title.

730

Synthesis and Evaluation of [18F]Cinacalcet for the Imaging of Parathyroid Hyperplasia

Anna L. Pees1, Esther Kooijman1, Robert C. Schuit1, Maria J. W. D. Vosjan2, Anton Engelsman3, Albert D. Windhorst1, Danielle J. Vugts1

1 Amsterdam UMC, VU University Medical Center, Department of Radiology and Nuclear Medicine, Amsterdam, Netherlands
2 BV Cyclotron VU, Amsterdam, Netherlands
3 Amsterdam UMC, Academic Medical Center, Department of Surgery, Amsterdam, Netherlands

Introduction

The parathyroid glands play an essential role in the regulation of blood calcium levels by secreting parathyroid hormone (PTH). In hyperparathyroidism, secretion of PTH is increased, leading to bone erosion and kidney stones. Surgical removal of the over-active glands is the standard of care. However, this requires precise pre-operative localization of the glands. To overcome limitations of the current localisation techniques our aim was to develop a new PET tracer based on cinacalcet, a drug binding to the calcium-sensing receptor of the parathyroid glands.[1]

Methods

[18F]Cinacalcet synthesis was carried out according to scheme 1. Commercially available 3-(3-chlorophenyl)propan-1-ol 1 was converted to tosylate 2 and reacted with tert-butyl (R)-(1-(naphthalen-1-yl)ethyl)carbamate 3 resulting in compound 4. The boronic acid functionality was obtained by Miyaura borylation and subsequent hydrolysis of the ester.[2] For the labelling of the resulting precursor 5 with fluorine-18, 5 was reacted in a copper-catalysed reaction with [18F]fluoroform.[3] After deprotection, purification by preparative HPLC (Gemini C18 column, MeCN/H2O/TFA 53:47:0.1) and formulation, [18F]cinacalcet was obtained in a solution ready for injection.

Results/Discussion

Compound 5 was obtained in an overall yield of 12%. 5 was reacted under various conditions with [18F]fluoroform. Under non-optimized conditions, compound 6 could be made in 17-61% radiochemical yield (determined by HPLC). The deprotection of the intermediate 6 resulting in [18F]cinacalcet proceeded smoothly with close to quantitative yields (97±1%, n=2, determined by HPLC). Purification of the product by preparative HPLC resulted in high radiochemical purity (>95%). After solvent exchange via a tC18 sep-pak cartridge, [18F]cinacalcet was obtained in 10% ethanolic saline solution with an overall RCY of 5.5±1.5% (n=3) and a molar activity of 42 GBq/µmol  in a total synthesis time of 1 hour. The product was stable in this solution for at least 2 hours.

Conclusions

A synthesis strategy for the potential PET tracer [18F]cinacalcet 7 was successfully developed, providing the product with an overall RCY and RCP suitable for animal studies. Experiments in healthy rats are scheduled to assess the viability of  [18F]cincalcet as PET tracer for localisation of the parathyroid glands.

Acknowledgment

The project is financially supported by The Netherlands Organization of Scientific Research (NWO) and BV Cyclotron VU.

References
[1] Bilezikian, J. P.,  Bandeira L., Khan, A., Cusano, N. E. 2018, 'Hyperparathyroidism', Lancet, 391, 168-178.
[2] Jiang, X., Chu, L., Qing, F. L., 2012, 'Copper-Catalyzed Oxidative Trifluoromethylation of Terminal Alkynes and Aryl Boronic Acids Using (Trifluoromethyl)trimethylsilane', J. Org. Chem., 77, 1251–1257.
[3] Van der Born, D., Sewing, C., Herscheid, J. D. M., Windhorst, A. D., Orru, R. V. A., Vugts, D. J., 2014, 'A Universal Procedure for the [18F]Trifluoromethylation of Aryl
Iodides and Aryl Boronic Acids with Highly Improved Specific Activity', Angew. Chemie Int. Ed., 53, 11046–11050.
Scheme 1
Synthesis of [18F]cinacalcet. Reaction conditions: a TsCl, DMAP, Et3N, DCM, 40 °C; b NaH, DMF, 35 °C; c B2pin2, Pd2(dba)3/PCy3, KOAc, dioxane, 95 °C; d NaIO4, NH4OAc, H2O/acetone; e [18F]CHF3, CuBr, KOtBu, Et3N·3HF, DMF; f HCl.
Keywords: [18F]cinacalcet, parathyroid hyperplasia, [18F]fluoroform
731

In Vivo Necrosis Imaging with Indium-111 Labeled 800CW

Marcus C. M. Stroet1, Erik de Blois1, Debra C. Stuurman1, Corrina M. A. de Ridder1, Joost Haeck1, Yann Seimbille1, Laura Mezzanotte1, Marion Hendriks - de Jong1, Clemens W. G. M. Lowik1, Kranthi Panth1

1 Erasmus MC, Radiology and Nuclear Medicine, Rotterdam, Netherlands

Introduction

Current clinical measurements for tumor treatment efficiency rely on changes in tumor morphology, which take time to become apparent and pose a physical and economic burden on the patient. However, there are no reliable clinical tools for detection of tumor necrosis. In previous research, we studied the necrosis avidity of cyanine-based fluorescent dyes to detect cell death in the tumor as a fast measure for treatment efficiency. We now demonstrate the application of radiolabeled 800CW, a commercially available and hydrophilic fluorescent dye, to image tumor necrosis in vivo.

Methods

We conjugated 800CW to a DOTA chelator via amine reactive crosslinking chemistry to yield DOTA-PEG4-800CW, a precursor for labeling 800CW with single photon emission computed tomography isotope indium-111. We developed effective and reliable procedures for preparation and analysis of [111In]In-DOTA-PEG4-800CW. We then investigated specific dead cell binding of [111In]In-DOTA-PEG4-800CW on dead or alive 4T1 cells in vitro, using both fluorescence and radioactivity as detection modalities. Finally, we investigated the binding of [111In]In-DOTA-PEG4-800CW to necrosis in a 4T1 tumor model in mice.

Results/Discussion

We successfully prepared a precursor and developed a reliable procedure for labeling 800CW with indium-111. We detected specific dead cell binding of [111In]In-DOTA-PEG4-800CW using both fluorescence and radioactivity. Albeit with a tumor uptake of 0.37 %ID/g at 6 hour post injection, we were able to image tumor necrosis with clear tumor to background ratios. Fluorescence and radioactivity in cryoslices from the dissected tumors were colocalized with tumor necrosis, confirmed with TUNEL staining.

Conclusions

800CW can be used to image tumor necrosis in vivo. Further research will elucidate the application of hydrophilic cyanine dyes as 800CW for the detection of necrosis caused by chemotherapy. This can provide valuable prognostic information in treatment of solid tumors.

References
[1] Xie B, Stammes MA, van Driel PBAA, et al 2015, Necrosis avid near infrared fluorescent cyanines for imaging cell death and their use to monitor therapeutic efficacy in mouse tumor models. Oncotarget 6:39036–39049. https://doi.org/10.18632/oncotarget.5498
In vitro and in vivo evaluation of necrosis binding of [111In]In-DOTA-PEG4-800CW

A) 4T1-Luc2 cells in a 12 well plate, cells in the two wells on the left killed with EtOH, cells in the two wells on the right kept alive, then treated with either [111In]In-DOTA-PEG4-800CW or [111In]In-DOTA-PEG4-NH2. B) representative SPECT image of a mouse 6 h.p.i. of [111In]In-DOTA-PEG4-800CW. C) Adjacent cryoslice from a dissected tumor from mouse treated with [111In]In-DOTA-PEG4-800CW. From left to right: autoradiography, fluorescence imaging and TUNEL dead cell staining.

Keywords: Necrosis imaging, SPECT, cyanine dyes
732

Validation of positron emission tomography tracers for the in vivo detection of reactive oxygen species

Edward C. T. Waters1, Friedrich Baark1, Thomas Eykyn1, Richard Southworth1

1 King's College London, Division of Imaging Sciences & Biomedical Engineering, London, United Kingdom

Introduction

Detecting changes in tissue redox status by PET imaging of ROS could enable earlier diagnosis of disease, drug toxicity or therapeutic response. While several ROS-sensing PET tracers have recently been postulated, none have been validated in terms of parallel independent biomarkers of oxidative stress or exclusion of the confounding effects of changes in tissue perfusion. We have developed a model of ROS generation in isolated perfused rat hearts which allows such validation and characterisation. Here, we demonstrate its utility in evaluating our pilot ROS sensing tracer; [18F]dihydroethidine.

Methods

The chemoselectivity of [18F] dihydroethidine to various ROS was first evaluated in vitro by incubation with different pro-oxidants and subsequent analysis by HPLC. Then, isolated perfused hearts from Male Wistar rats (250-300g), were excised and perfused in the Langendorff mode with menadione (0, 10 and 50μM) to invoke intracellular superoxide generation, or antimycin A   (0, 30 and 100nM) to invoke mitochondrial superoxide generation. The model was validated with measurements of total tissue glutathione concentration and PKG1α oxidation, then used to assess our ROS sensitive tracer. [18F] dihydroethidine (3-5 MBq) was infused into the arterial perfusion line at a constant rate, and the rate of cardiac tracer retention was determined using our custom built gamma detection apparatus.

Results/Discussion

[18F] dihydroethidine was synthesised in a non-decay corrected radiochemical yield of 12 ±3% (n=10) with a molar activity of >74 GBq.µmol-1. The logD7.4 was 1.58 ± 0.11 (n=6) for the neutral tracer and -0.22 ± 0.04 (n=6) for its oxidised form, which would allow tissue penetration and ROS-dependent trapping respectively. The tracer showed selectivity for both superoxide (25% oxidation after 5 minutes and 50% oxidation after 1 hour n = 3) and hydroxyl radicals (20% oxidation after 5 minutes and 1 hour n=3).

Menadione treatment depleted GSH to 67.1 ±1.5% (n=3) of control, and elevated PKG1α  oxidation from 11.0 ±0.1% to 68.1 ±0.2%. Under these conditions of ROS generation, the rate of cardiac [18F] dihydroethidine uptake increased 77.7 ±6.3% (n=4) when compared to control.

Conclusions

We have used a validated model of ROS generation to demonstrate the superoxide-dependent cardiac trapping of [18F] dihydroethidine. As perfusate wasdelivered to the isolated heart by constant flow, this increased accumulation can be unequivocally attributed to validated ROS generation. This is not the case for current in vivo methods of ROS generation that do not account for changes in tissue perfusion.

AcknowledgmentThis work was funded by the EPSRC, the EPSRC centre for medical engineering and the BHF.
Figure 1
A LogD values of both reduced (grey) and oxidised (black) [18F]DHE. (mean ± SD, n=6). B Selectivity of [18F]DHE for ROS (mean ± SD, n=3).
Figure 2
A Custom triple γ-detector array measuring radioactivity in an isolated perfused rat heart. B Rate of uptake of [18F]dihydroethidine in control and menadione perfused hearts (n=4, ± SD, *=P<0.05).
Keywords: ROS, PET, Cardiovascular, Perfusion
733

Microwaves Allow Fast 52Mn Radiolabelling of Porphyrins: Applications in Cell and Liposome Labelling

Peter J. Gawne1, Sara M. Pinto2, Karin M. Nielsen3, Mariette M. Pereira2, Rafael T. M. de Rosales1

1 King's College London, School of Biomedical Engineering & Imaging Sciences, London, United Kingdom
2 University of Coimbra, Coimbra Chemistry Center, Coimbra, Portugal
3 Technical University of Denmark, The Hevesy Lab, Roskilde, Denmark

Introduction

Manganese porphyrins have several therapeutic/imaging applications. Their superoxide dismutase activity and paramagnetism makes them efficient tumour chemo/radiosensitisers (in clinical trials),[1] and MRI contrast agents,[2] respectively. The affinity of porphyrins for lipid bilayers also makes them candidates for cell/liposome labeling. We hypothesised that metallation with the PET radionuclide 52Mn (t1/2 = 5.6d) would allow long-term in vivo studies of Mn-porphyrins, as well as a method to label and track cells/liposomes, but methods for fast and efficient radiolabelling are lacking.

Methods

52MnCl2 and porphyrins 14 (Fig. 1A) were produced as previously reported.[3,4] Porphyrins 14 were added to neutralised 52MnCl2 and heated at 165oC for 1h (MW) at a ligand concentration of 0.6 mM. These conditions were compared with standard heating at 70oC over 24h. Radiolabelling yields were evaluated by iTLC/TLC and HPLC, and the resulting 52Mn-porphyrins characterised by comparison with their non-radioactive 55Mn counterparts and logP measurements. Following this, 52Mn-(1-4) were used to radiolabel liposomes by incubation at 50oC for 30min and purified by size-exclusion chromatography. 52Mn-(1-2) were taken forward to label MDA-MB-231 cells in vitro and compared with a previously reported direct-labelling agent, 52Mn-oxine.[5]

Results/Discussion

MW radiosynthesis allowed fast (1h) and efficient radiolabelling with >95% radiochemical yields (RCY) with ligands 1, 2 & 4, and 85 ± 7% for 52Mn-(3). This increased to 95 ± 2% RCY by using a ligand 3 concentration of 0.7 mM (Fig. 1B). Conversely, non-MW heating at 70oC for 1h resulted in ca. 25% RCY for 52Mn-(2 & 4), with no conversion for 52Mn-(1 & 3) (Fig. 1B & C). After 24h at 70oC, the RCYs increased, without reaching completion. Formation of the 52Mn-complexes were confirmed with radio-HPLC (52Mn-(1) trace in Fig 1D), iTLC/TLC (Fig. 1E) and logP (Fig 1F). 52Mn-(1-3) labelled liposomes with high efficiencies of 78 ± 3%, 86 ± 2% and 74 ± 7%, respectively (Fig. 2A). 52Mn-(1-2) radiolabelled MDA-MB-231 cells with 10 ± 7%LE and 12 ± 1%LE, respectively (Fig. 2C) whereas 52Mn-oxine (Fig. 2B) had 42 ± 2% LE. However, the cellular retention of 52Mn after 24h via 52Mn-oxine radiolabelling was significantly lower (14 ± 1%), compared with 52Mn-(1) (32 ± 5%) and 52Mn-(2) (45 ± 4%) (Fig. 2D).

Conclusions

In contrast to standard methods, MW heating allows the fast synthesis of 52Mn-porphyrins with >95% radiochemical yields that avoid purification. This technique can be exploited for the in vivo imaging of Mn-porphyrin therapeutics, as well as for the accurate in vivo quantification of Mn-porphyrin MRI agents. 52Mn-porphyrins also show promising cell/liposome labelling properties, with improved 52Mn cell retention over direct labelling methods.

AcknowledgmentThe authors would also like to acknowledge Jesper Fonslet for his previous productions of Manganese-52. 
References
[1] Batinic-Haberle I. et al., Redox Biology. 2019, 25, 101139
[2] Calvete M. et al., Coord Chem Rev. 2017, 333, 82-107
[3] Fonslet J. et al., App Rad Isop. 2017, 121, 38-43
[4] Monteiro C. et al., Tetrahedron, 2008, 64, 5132-5138
[5] Gawne P. et al., Dalton Trans. 2018, 47, 9283-9293
Fig. 1: Summary of the radiolabelling and characterisation of the radiomanganese porphyrins.
A) Structures of the porphyrin ligands 14, along with the reaction scheme for the microwave synthesis method. B) Comparison of the radiochemical yields achieved over 24 h and C) after 1 h for the microwave synthesis method compared to that achieved with labelling at 70 oC for the porphyrins. D) Radio-HPLC trace (top) of 52Mn-(1), UV trace of non-radioactive Mn-(1) (bottom). Note the presence of isomers for the porphyrin complex. E) iTLC/TLC and F) Log P characterisation of the 52Mn-porphyrin complexes. All Error bars represent mean ± SD (n = 3) except for the 70oC RCY (n = 1).
Fig. 2: Summary of the liposome and cell radiolabelling properties of the radiomanganese porphyrins.
A) Graph showing the liposome labelling properties of each of the 52Mn-porphyins. B) Structure of 52Mn-oxine. C) Graph showing the % cell labelling of 52Mn-(1) and 52Mn-(2) with MDA MB-231 cells compared with 52Mn-oxine and unchelated 52Mn. D) Graph showing the cellular retention of 52Mn in MDA MB-231 cells labelled with 52Mn-oxine, 52Mn-(1) and 52Mn-(2) after 24 h. All Error bars represent mean ± SD (n = 3)
Keywords: PET, Porphyrins, manganese-52, liposome labelling, cell labelling
734

Nanoscale microfluidic reactions: towards near-stoichiometric carbon-11 radiolabelling for PET

Fraser G. Edgar1, 2, Salvatore Bongarzone2, Antony Gee2, Philip Miller1

1 Imperial College London, Department of Chemistry, London, United Kingdom
2 King's College London, School of Biomedical Engineering & Imaging Sciences, London, United Kingdom

Introduction

Carbon-11 is a radionuclide commonly used to radiolabel molecular imaging probes for PET. The most common route for carbon-11 incorporation is via 11C-methylation using [11C]methyl iodide ([11C]CH3I).1 Microfluidics concerns the manipulation of small volumes (μL - pL) of liquids within discrete microchannels. The aim of this work is to develop a droplet-based microfluidic approach to facilitate rapid radiolabelling on a scale that will circumvent post-radiolabelling purification. As proof of principle a dual-loop system was developed for rapid droplet-based (40 μL) 11C-methylation reactions.2

Methods

[11C]CO2 was produced using a Siemens RDS 112 cyclotron by 11 MeV proton bombardment of nitrogen-14 (+ 1% O2) gas. The [11C]CO2 was then transferred in a stream of helium to a GE TRCAERLab® FX MeI module where [11C]CH3I was then produced by gas phase conversion.
A simple microtube set-up has been established using two 2 mL stainless steel HPLC loops connected to an E&Z modular lab for valve control with PTFE tubing (figure 1).
Prior to reaction the HPLC loops were charged with 40 μL of N-benzylmethylamine in DMSO to facilitate trapping and reaction. The [11C]CH3I was loaded onto each loop in a stream of helium (190°C, 30 mL/min). Following trapping the loop was heated, followed by cooling and elution with H2O. Activities were recorded and an alliquot submitted for radio-HPLC analysis.

Results/Discussion

Trapping of [11C]CH3I at room temperature was found to be consistently high, with achievable activity yields of 55 % (n = 5) without the need for base and with a heating step of only one minute. Heating for one minute at 150°C gave the highest achievable RCP as determined by radio-HPLC of 97 % (figure 2). Radiochemical purities varied with heating and concentration, lower temperatures gave lower RCPs (75 - 95 %), and decreasing the droplet concentration from 10.3 mM to 2.06 mM decreased the achievable RCP to 91 %.
This variance exemplifies the limit of the in-loop methodology for near-stoichiometric reactions as the volume of the droplet must be kept sufficiently high to ensure trapping of the [11C]CH3I, while retaining a high enough substrate concentration for efficient radiolabelling.
Current work is focusing on the radiolabelling of clinically-relevant 11C-radiopharmaceuticals, primarily [11C]methionine, assessing the current set-up suitability for S-methylation vs N-methylation.

Conclusions

In conclusion, this experimental set-up was found to be suitable for the trapping and subsequent radiolabelling with [11C]CH3I. Trapping of the total radioactivity was high and radiolabelling could be achieved with high chemical and radiochemical yields within 23 mins (n = 5) of EOB. In addition, the volume of DMSO used is below the residual solvent limit for pharmaceuticals. Future work will focus on the implementation of cryogenic trapping.

Acknowledgment

Fraser Graeme Edgar is funded through the Centre for Doctoral Training awarded by EPSRC, grant number; EP/L015226/1.

References
[1] Dahl, K., Halldin, M., Schou, M., 2017, 'New methodologies for the preparation of carbon-11 labeled radiopharmaceuticals', Clin. Transl. Imaging., 5, 275-289, New York City: Springer
[2] Downey, J., Bongarzone, S., Hader, S., Gee, AD., 2018, 'In-loop flow [11C]CO2 fixation and radiosynthesis of N,N'-[11C]dibenzylurea', J. Label. Compd. Radiopharm., 61, 263-271, New Jersey: Wiley
Figure 1
Schematic of in-loop set for 11C-methylations.
Figure 2
Radiochromatogram of crude radiolabelled product. Region 1 = [11C]MeOH 0.5 %, region 2 = [11C]CH3I 2 %, and region 3 = [11C]N,N-benzyldimethylamine 97%.
Keywords: carbon-11, 11C-methylation, In-loop, Microfluidics
735

Transport into the central nervous system of [18F]FLT and [18F]Fludarabine for brain tumors

Mihaela-Liliana Tintas1, Méziane Ibazizène1, Stéphane Guillouet1, Fabien Fillesoye1, Cyril Papamicaël2, Vincent Levacher2, Louisa Barré1, Fabienne Gourand1

1 Normandie Univ, UNICAEN, CEA, CNRS, ISTCT/LDM-TEP group,, Caen, France
2 Normandie Univ, COBRA, UMR 6014 et FR 3038, Univ Rouen, INSA Rouen; CNRS, IRCOF, Mont Saint Aignan, France

Introduction

[18F]FLT is widely used as a proliferation radiotracer for oncological PET studies for the diagnosis of high-grade gliomas and the follow-up of treatments.1 In contrast, low-grade brain gliomas are poorly visualised and the vectorisation of [18F]FLT through the BBB could provide the opportunity to detect these tumours at an early stage. [18F]Fludarabine is a novel specific radiotracer for non-Hodgkin’s lymphoma 2,3 and its vectorisation has been undertaken for early primary cerebral lymphoma imaging.

Methods

The transport into the central nervous system of both [18F]radiotracers ([18F]FLT and [18F]Fludarabine) has been studied thanks to a chemical delivery system (CDS) based on a redox system 1,4 dihydroquinoline/ quinolinium salt. A lipophilic carrier based on 1,4-dihydroquinoline structure was linked to the radiotracer and  after BBB passage, an oxidation followed by an enzymatic cleavage will lead to the release of the PET probe. To determine the best carrier, various sets of [11C]CDS-FLT  and  [11C]CDS-Fludarabine were prepared and evaluated in vivo  into rats.4 Further studies were to develop the radiolabelling of the entities CDS-[18F]radiotracer in order to  evaluate them in appropriate animal models.

Results/Discussion

Various parameters (base, solvent, temperature and time  reaction) have been studied i) to optimize the sensitive coupling reaction of the quinolinium salt  with  the [18F]radiotracer  and ii) to avoid some side reactions due to our high dilution conditions. The reduction step has been conducted with BNAH and has led to the corresponding CDS-[18F]radiotracer. The radiosynthesis was performed as a one-pot two-step procedure and after HPLC purification, the CDS-[18F]radiotracer  have been obtained with  a percentage  ranging from 45 to 65 %  (based on HPLC profiles).

Conclusions

The radiosynthesis of CDS-[18F]radiotracer  were successfully accomplished and  in vivo evaluation will be undertaken to study the accumulation of [18F]FLT/[18F]Fludarabine after their  release into the brain.

AcknowledgmentThis study was supported by a grant from CEA (Commissariat à l’Energie Atomique et aux Energies Alternatives), Labex IRON (ANR-11 LABX-0018-01), INSA-Rouen, Rouen University, CNRS, Labex SynOrg (ANR-11-LABX-0029), Région Normandie.
References
[1] Ullrich R et al. Clin. Cancer Res. 2008, 14, 2049-2055.
[2] Barré L et al. J. Nucl. Med. 2018, 59 (9), 1380-1385.
[3] Barré L et al. Theranostics. 2018, 16, 4563-4573.
[4] Gourand F et al. ACS Chem.Neurosci. 2017, 8 (11), 2457–2467.
Keywords: Brain-targeted delivery system, brain tumors, chemical delivery system
736

Tripodal N-centred Phosphine Ligands: Towards a Novel Donor Set for 99mTc Radiopharmaceutical Formulation

Saul Cooper1, 2, Thomas Yue1, Thomas Eykyn2, Philip Miller1, Michelle Ma2, Nicholas Long1

1 Imperial College London, Department of Chemistry, London, United Kingdom
2 King's College London, School of Biomedical Engineering and Imaging Sciences, London, United Kingdom

Introduction

N-triphos derivatives (NP3R, R = alkyl, aryl), and asymmetric variants (NP2RXR’, R’ = alkyl, aryl, X  = OH, NR2, NRR’) represent an underexplored class of tuneable, tripodal ligands in the coordination chemistry of Re and Tc for biomedical applications.1 Importantly, these ligands have the potential to form well-defined and isomer-free coordination spheres.2,3 Our aim has been to prepare a series of model complexes containing these tripodal phosphine ligands, and to assess their structure and reactivity prior to attempted translation to 99mTc radiopharmaceutical formulation.

Methods

Several NP2RXR’ derivatives are readily synthesised via phosphorus-based Mannich reactions.4 This reaction facilitates the introduction of functionalised secondary phosphines into an appropriate ligand scaffold. The reactivity between these ligands and a number of translationally relevant non-radioactive Re(I) and Re(V) precursors, including [NEt4][ReBr3(CO)3], ReOCl3(PPh3)2,[NBu4][ReOCl4] and [ReIO2(PPh3)2], has been investigated. The resultant complexes have been fully characterised using standard techniques (NMR, IR, mass spectrometry, and X-ray crystallography). Studies on the reactivity of these ligands with [99Tc]-[NBu4][TcOCl4] have also been undertaken to compare the two metal congeners, and analysed by NMR spectroscopy.

Results/Discussion

NP3Ph has been shown to form kinetically stable complexes exhibiting both bidentate and tridentate coordination to the fac-[Re(CO)3]+ fragment. However, these symmetric ligands exhibit only bidentate coordination in Re(V) complexes. The use of the asymmetric NP2PhOHAr ligand has been shown to facilitate tridentate coordination towards both fac-[Re(CO)3]+ and [ReO]3+ units. The latter case provides the basis for a novel, reactive metal fragment for stabilisation of the [ReO]3+ core; [ReOCl23-NP2PhOAr)] (Figure 1). The reactivity of this species towards further functionalisation with π-donors has also been explored. Furthermore, these chelators have the potential to be chemically modified to incorporate additional functionality that would enable them to act as bifunctional chelators. Analogous species to the Re(V) complexes can also be synthesised with 99Tc(V) and these complexes exhibit highly similar structural properties.

Conclusions

Non-radioactive rhenium studies have illustrated the potential of these tripodal, N-centred phosphine ligands towards the stabilisation of Re cores in both +1 and +5 oxidation states in a series of model complexes. The synthesis of M(V) species can also be extended to 99Tc chemistry. We are now looking at applying these ligands in the formulation of well-defined 99mTc complexes and hope to present this data in due course.

Acknowledgment

This project is funded as part of the EPSRC Centre for Doctoral Training in Medical Imaging at Imperial College London and King’s College London.

References
[1] Liu, S., 2008, Adv. Drug Deliv. Rev.60, 1347–1370.
[2] Phanopoulos, A., White, A. J. P., Long, N. J. and Miller, P. W., 2016, Dalton Trans.45, 5536–5548.
[3] Apps, S. L., White, A. J. P., Miller, P. W., and Long, N. J., 2018, Dalton Trans.47, 11386–11396.
[4] Cao, B., Elsegood, M. R. J., Lastra-Calvo, N., and Smith, M. B., 2017, J. Organomet. Chem.853, 159–167.
Figure 1
The NP2PhOHAr ligand forms the reactive metal fragment [ReOCl2(NP2PhOAr)] upon reaction with suitable Re(V) precursors in basic media. It can be further functionalised with appropriate bidentate ligands to form a kinetically stable complex.
Keywords: SPECT, Technetium-99, Rhenium, Phosphine
737

Room Temperature Al18F Labeling of 2-Aminomethylpiperidine-Based Chelators for PET Imaging

Lisa Russelli1, Jonathan Martinelli2, Francesco De Rose1, Sybille Reder1, Michael Herz1, Markus Schwaiger1, Wolfgang A. Weber1, Lorenzo Tei2, Calogero D'Alessandria1

1 Klinikum rechts der Isar der TUM, Department of Nuclear medicine, Munich, Germany
2 Università del Piemonte Orientale, Department of Science and Technological Innovation, Alessandria, Italy

Introduction

The use of antibody-derived imaging probes for Positron emission tomography (PET) is constantly expanding. Due to its short half-life, 18F matches the biological half-life of small heat sensitive biomolecules such as Fabs, nanobodies, scFv, for which labeling with AlF-18 is a promising approach1-2. To this purpose, we have designed and characterized three chelators based on the structure of 2-aminomethylpiperidine with acetic and/or hydroxybenzyl pendant arms (2-AMPTA, NHB-2-AMPDA and 2-AMPDA-HB), and tested for AlF-18 labeling and for their stability in physiological conditions.

Methods

Three different AMP-based chelators (2-AMPDA-HB/NHB-2-AMPDA/2-AMPTA) were designed, synthetized and characterized by HPLC-MS and NMR spectroscopy. AlF-18 labeling reactions were performed at different pH and temperature (rt, 37, 80°C) and compared to labelling at the same temperatures of p-SCN-Bn-NOTA chelator, as reference. After purification, all the products were analyzed by radio-TLC and radio-HPLC and the pH effect was investigated from 4 up to 6.5. In vitro stability of the complexes was investigated at 10, 30, 60, 120 and 240min via incubation in human serum, PBS and 0.9% NaCl solutions. In vivo stability was studied via dynamic PET/CT scan and ex vivo biodistribution on healthy nude mice injected with the Al18F-complex among the three chelators showing highest stability in vitro.

Results/Discussion

For all chelators a radiolabeling efficiency between 55 and 81% was obtained at pH 5 and room temperature (55% 2-AMPDA-HB, 69% NHB-2-AMPDA and 81% 2-AMPTA), with similar results obtained at 37 and 80°C where the RCY for 2-AMPDA-HB reached 75%. As expected, low RCYs were obtained with the p-SCN-Bn-NOTA at the same temperatures (0.96% at T-room, 3.72% at 37°C and 25.3% at 80°C). The AMP-based chelators are able to complex {Al18F}2+ with good RCYs also at higher pH, with a RCY of ~50% at pH 6.5. High stability in human serum was measured for the compound Al18F(2-AMPDA-HB)]-, with a 90% of AlF-18 complexed up to 120 min and 68% up to 240 min. Al18F(2-AMPDA-HB)]- complex presented in vivo fast blood clearance, hepatobiliary and renal excretion, and based on biodistribution results, the labelled complex showed high stability in vivo as confirmed by the low 18F accumulation measured in femur (%ID/g: 1.63±0.73%) and other organs at 2 h p.i..

Conclusions

Good results have been obtained in terms of RCY at T-room and, notably, high stability in HS (>90%) measured at 120min3, compared to other chelators for Al18F labeling4. The [Al18F(2-AMPDA-HB)]-  compound showed good stability in vivo, with a low accumulation of 18F in bones. These results prompted us towards the synthesis of a bifunctional 2-AMPDA-HB to facilitate the fluorination of heat-sensitive biomolecules and their in vivo applications.

References
[1] McBride,WJ, Sharkey, RM, Karacay, H, D’Souza, CA, Rossi, EA, Laverman, P, Chang, CH, Boerman, OC, Goldenberg, DM 2009, 'A Novel Method of 18F Radiolabeling for PET', J. Nucl. Med., 50, 991–998
[2] Cleeren, F, Lecina, J, Bridoux, J, Devoogdt, N, Tshibangu, T, Xavier, C, Bormans, G 2018, 'Direct fluorine-18 labeling of heat-sensitive biomolecules for positron emission tomography imaging using the Al18F-RESCA method', Nat. Protoc., 13, 2330–2347
[3] Russelli, L, Martinelli, J, De Rose, F, Reder, S, Herz, M, Schwaiger, M, Weber, WA, Tei, L and D'Alessandria, C 2020, 'Room Temperature Al18F Labeling of 2-Aminomethylpiperidine‐Based Chelators for PET Imaging', ChemMedChem
[4] Cleeren, F, Lecina, J, Billaud, EMF, Ahamed, M, Verbruggen, A, Bormans, GM 2016 'New Chelators for Low Temperature Al18F‑Labeling of Biomolecules' Bioconjugate Chem., 27, 3, 790-798
Figure 1
Radiochemical yields (±SD) at pH 5 of Al18F-AMP-derivatives and Al18F-p-SCN-Bn-NOTA at different temperatures analyzed via radio-TLC. On X-axis are reported the reaction temperatures, while on the Y-axis the radiochemical yields as a percentage (n=3).
Figure 2
Maximum Image Projections (MIPs) obtained selecting frames at different time points during dynamic PET-CT scans. A hepatobiliary and renal excretion is clearly visible. Scale bar: 0–20%ID/g.
Keywords: AlF-18, Radiofluorination, Polidentate chelators, PET imaging
739

Production of [68Ga]Ga-PSMA-11: The manual kit-based approach versus an automated synthesis method

Janke Kleynhans1, 2, 3, Sietske Rubow2, Jan Rijn Zeevaart4, 3, 5, Jannie le Roux2, 6, Thomas Ebenhan1, 3

1 University of Pretoria, Nuclear Medicine, Pretoria, South Africa
2 Stellenbosch University, Nuclear Medicine Division, Tygerberg, South Africa
3 Nuclear Medicine Research Infrastructure, Preclinical Imaging Faclity, Pretoria, South Africa
4 North-West University, Preclinical Drug Development Platform, Potchefstrooom, South Africa
5 Nuclear Energy Corporation of South Africa, Radiochemistry, Pelindaba, South Africa
6 Nuclear Medicine Research Infrastructure, Node for Infection Imaging, Tygerberg, South Africa

Introduction

The growing demand for Gallium-68 -labeled radiopharmaceuticals necessitated radiosynthesis methods which limit radiation exposure [1,2]. This analysis evaluates two methods for gallium-68 radiolabeling of the prostate-specific membrane antigen targeting agent (PSMA-11). [68Ga]Ga-PSMA-11 PET/CT used now widely used for imaging all types of PSMA-expressing prostate cancer . An automated synthesis method is compared with a single vial kit-based radiolabelling process to provide an analysis of the advantages and disadvantages of each method.

Methods

An automated, cassette-based GMP-complaint radiosynthesis module (Scintomics GmbH and ABX GmbH, Germany) was used to produce [68Ga]GA-PSMA-11 in one method. In the other method aseptically produced PSMA-11 kits (Nuclear Energy Corporation of South Africa) were labelled manually by protocols and analysis methods as described before [3,4]. Ga-68-activity was obtained from commercially available 68Ge/68Ga generators from (iThemba LABS, South Africa). A retrospective data analysis was performed for each method (N=40 syntheses), comparing the quality of the final product (radionuclidic purity, radiochemical purity, sterility, physiological acceptability), product yield (molar activity, total yield, the robustness of synthesis), radiation burden on operators and economic considerations.

Results/Discussion

Neither of the generators demonstrated long-lived isotope breakthrough (germanium-68) with the end products demonstrating the correct radionuclidic identity by half-life measured and no residual germanium-68 present the next day. Radiochemical purity (≥ 95%) adhered to release criteria (EANM guidelines) for both methods. Radiochemical yields did not differ significantly. Products both adhered to release criteria for pH of the final product, membrane filter integrity, presence of bacterial endotoxins (<10 EU/ml), residual ethanol content and bench-top stability (> 3 h).
Statistically significant differences were shown (Table 1) for synthesis time, molar activity concentration, activity yield and final product activity concentration. However, none of these differences resulted in a product that did not adhere to release criteria. The differences in radiation exposure per synthesis and the cost of synthesis are parameters that could influence operations the choice of method.

Conclusions

Both methods are reliable alternatives to the manual synthesis of [68Ga]Ga-PSMA-11. Given sufficient infrastructure and cost considerations, an automated, cassette-based radiosynthesis will provide a GMP-compliant radiopharmaceutical. The kit-based method is a useful, inexpensive alternative which produces a radiopharmaceutical with excellent quality, however, adequate radiation protection measures must be in place.

Acknowledgment

The authors acknowledge the HPLC analyses performed by Ms Biljana Marjanovic-Painter. The staff of the Nuclear Medicine Department of Steve Biko Academic Hospital are thanked for their assistance in day-to-day operations.

References
[1] Boschi, S, Lodi, F, Malizia, C, Cicoria, G, Marengo, M 2012, Automation synthesis modules review. Applied radiation and isotopes, 76:38-45.
[2] Heidari, P, Szretter, A, Rushford, LE, Stevens, M, Collier, L, Sore, J, Hooker, J, Mahmood, U 2016, Design, construction and testing of a low-cost automated 68Gallium-labelling synthesis unit for clinical use. American Journal of Nuclear Medicine and Molecular Imaging, 6(3):176-184.
[3] Ebenhan, T, Vorster, M, Marjanovic-Painter, B, Wagener, J, Suthiram, J et al. 2015, Development of a single vial kit solution for radiolabeling of 68Ga-DKFZ-PSMA-11 and its performance in prostate cancer patients. Molecules, 20:14860-14878.
[4] Le Roux, J, Rubow, S, Ebenhan, T, Wagener, C 2019, An automated synthesis method for 68Ga-labelled ubiquicidin 29-4. Journal of Radioanalytical and Nuclear Chemistry. DOI: 10.1007/s10967-019-06910-1.
Table 1
Statistically significant differences in outcomes between the manual kit-based approach and the automated synthesis method
Keywords: positron emission tomography, Good Manufacturing Practice, 68Ga-DKFZ-PSMA-11